【作者】 李彩霞；

【导师】 张学光；

【作者基本信息】 苏州大学 ， 血液学， 2004， 博士

【摘要】 多发性骨髓瘤(multiple myeloma，MM)是浆细胞在骨髓中呈恶性克隆增殖性疾病。与其它血液肿瘤不同，MM细胞起源于生发中心的记忆B细胞或前浆细胞，选择性归巢、定居在骨髓；到疾病的晚期，MM细胞可以播散至外周形成浆细胞白血病。而MM细胞的归巢及定位能促进MM细胞的生长、生存和迁移，从而导致病理性骨损害、骨折等一系列临床症状。迄今为止，MM仍然是一种不可治愈的疾病。研究发现，骨髓微环境中分泌的一些生物因子与表达在MM细胞上的趋化因子、黏附分子等受体相互作用，在MM细胞迁移、归巢并长期生存在骨髓过程中发挥至关重要作用。寻求以骨髓中的MM细胞生长及生存机制的靶向治疗一直是人们研究的焦点；因此深入研究MM细胞生长、生存及归巢机制以及一些促进MM细胞生长、生存及迁移的生物因子，具有重要的理论和潜在临床应用价值。 鉴此，我们采用细胞生物学和分子生物学技术，以人多发性骨髓瘤为研究对象，围绕基质细胞衍生因子SDF-1与其受体CXCR4在MM细胞迁移、归巢及其生长中的生物学作用，进行了下述三个方面的研究。 一、人多发性骨髓瘤患者血浆SDF-1的含量及MM细胞CXCR4的表达和作用SDF一1/C XCR4在人多发性骨髓瘤的表达及其生物学功能的研究中文摘要1、采用免疫荧光标记+流式细胞仪以及R丁一PCR方法检测了25例MM患 者的MM细胞和依赖工L一6生长的MM细胞株:XGI、XGZ、XG6和XG7 的CXCR4表达:EL工SA方法检测MM患者血浆SDF一1和工L一6的含量; 采用体外微孔隔离室进行体外SDF一诱导的迁移运动实验。结果发 现:①新鲜咖细胞及MM细胞株不同程度的表达功能性CxCR4[(42.7 士17，3)%〕，.其表达水平与体外对SDF一1迁移能力「(23.2士1.08) %」密切相关(P<0.01=;②MM患者骨髓血浆SDF几表达水平显著高 于正常人骨髓血浆表达水平〔(3489.23士651.63)pg/ml，(2818.57 士597.79) pg/ml，P<0.05二;外周血浆SDF一1表达水平低于正常 人外周血浆SDF一1的表达水平「(1973土133)pg/ml，(233吸.857士 574.923)，P二O;062〕;③浆细胞白血病患者外周血SDF一1含量 4097.143士680.71，显著高于正常人外周血SDF一l的含量(P<0.01)， 由此首次证实sDF一1/CXCR4在MM患者体内异常表达，且与枷细胞 的迁移运动相关。2、进一步研究证实，SDF一1/CXCR4相互作用可上调MM细胞表达粘附分 子ICAM一1(CD54)，工CAM一1的表达和可溶性ICAM一1的产生与CXCR4 表达有一定相关性;表明SDF一1/CXCR4对介导粘附分子的调节效应 在MM细胞的生物行为中起重要的作用。3、通过体外细胞培养观察，发现SDF一具有刺激XGI、XGZ细胞增殖的 作用，但其作用效果明显低于工L一6，P<0.01;EL工SA方法检测XGs 细胞培养上清及刚患者血浆中IL一6的表达水平结果显示:XGs细胞 经SDF且诱导培养后，上清中工L一6含量较培养前增加;60%MM患者 血浆IL一6含量较正常人显著增高;通过相关性分析表明，MM患者血 浆SDF一含量与工L一6含量正相关。因此，SDF~1/CXCR4相互作用， 可能通过促进自分泌和旁分泌IL一6两条途径调节MM的生长和增殖。4、通过免疫沉淀+Western blot分析表明，SDF一1/CXCR4作用的信号转 导途径是通过活化毗PK，而与STAT3无关。5、临床资料相关性分析发现，SDF一1/CXCR4在MM体内的异常表达与MM SDF一1/C xCR4在人多发性骨髓瘤的表达及其生物学功能的研究中文摘要 的临床症状、分期和免疫球蛋白分型等无关，但SDF--1/CXCR4在PCL 均显著异常增高，提示SDF一1/CXCR4的异常高表达可能与MM的疾病 进展、预后不良有关。 二、ICOS/GL50信号转导在MM细胞生物学行为中的作用 在上述SOF一1/CxCR4在枷作用的工作基础以及我们成功克隆和表达了-ICOS/GL50该对新型共刺激分子、构建了相应的的转基因细胞的基础上，采用细胞生物学和分子生物学技术研究率先发现: 1、!cos/GL50不同程度的异常表达或共表达在朋细胞 RT一PCR结果表明，从XGI、XGZ细胞和纯化的新鲜MM细胞的mRNA 中均能扩增出ICOS和GL50的特异性条带。免疫荧光标记和流式细胞 术分析结果表明，20例MM患者骨髓及外周血原始，幼稚浆细胞上表 达不同程度的GLSO分子，其中3例MM细胞共表达ICOS/GLSO:XGI、XGZ 细胞表面均异常表达JCOS分子，而XG6、XG7不表达ICOS分子。XGZ同 时中等程度表达GLSO分子。 2、COS/GLSO相互作用能够上调MM细胞功能性CXCR4的表达 工COS/GLSO相互作用可上调XGZ细胞CXCR4和CD54的表达，以作用后24h 最显著，而不表达工COS/GLSO的XG7细胞则无此变化。进一步体外迁移实验 证实ICOS/GLSO介导删细胞CXCR4的上调表达与SDF月诱导的迁移功能有 关。由此表明，工COS/GLSO分子的异常表达及其通过SDF一1/C XCR4途经调节 MM患者肿瘤细胞的生物学行为中具有重要作用。 三、s。「一1/CxcR4在人多发性骨髓瘤造血干细胞动员中的作用 目前，大量的临床资料己经表明，大剂量化疗联合造血干细胞移植己使更多还原

【Abstract】 Multiple myeloma(MM) is a monoclonal expansion of malignant cells with a plasmablast-plasma cell morphology that is almost exclusively localized to the bone marrow, except at the final stages of disease, when they proliferate in the extramedullary area. MM cells appear to be derived from a pst germinal centre B cell as they express somatically mutated Ig heavy chain genes. The mechanisms of the selective homing of MM cells to the bone marrow compartment are poorly understood. It is possible that the bone marrow localization reflects the expression of chemotactic receptors on MM cells that directs their migration to the bone marrow.The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 contribute to stem cell homing and play a role in trafficking of leukemic cells. In this study we have investigated expression and biological behavior of SDF-1/CXCR4 in MM-derived cell lines and primary MM cells.Part I. Plasma Levels of SDF-1 and Expression of SDF-1 Receptor onMultiply Myeloma Cells of Human Multiply Myeloma1. FACS and RT-PCR analysis was used to study the expression of CXCR4 and ICAM-1(CD54) on the surface of MM cells from 4 IL-6 dependant cell lines (XG1.XG2,XG6 and XG7) and 25 freshly isolated tumor samples from patients with diagnosed MM. Mononuclear cells were purified by positive selection of magnetical and FACS sorting. Chemotaxis assay through transwell bore polycaronate and ELISA assay were employed to monitor the SDF-1, IL-6, and sICAM-1 levels. We found that 瓼resh MM cells and MM cell lines expressed various levels of functional CXCR4ranging from 23.1% to 77.7%,which was correlated with the in vitro migration ability of MM cells[ (23.2?.08) %, PO.01]; DSDF-1 levels in the bone marrow(BM) of MM patients were significantly higher than the those of healthy persons [(3489.23?51.63)pg/ml, (2818.57?97.79)pg/mL P<0,05 = ; but plasma levels of SDF-1 in peripheral blood of MM patients were lower than those of healthy persons[(1973 ?133)pg/ml, (2334.857 ?574.92), P=0.062]; ㏄lasma levels of PCL(4097.14 ?80.71) were significantly higher than those of healthy persons, P< 0.01. The results firstly demonstrated abnormal expression of SDF-1 and its receptor CXCR4 on Human MM cells, which is closely correlated with the migration of MM cells.2. Furthermore, we discovered that SDF-1 could up-regulate the expression of ICAM-1 on MM cells; the plasma level of soluble ICAM-1 was correlated with the expression of CXCR4 on MM cells. These findings suggested that SDF-1/CXCR4 axis play a key role on the trafficking of MM cells via mediating the effect of adhesion molecules.3. We also found that SDF-1 could promote long-term proliferation of myeloma cells besides inducing migration of myeloma cells as IL-6 did. An up-regulation of autocrine IL-6(from 5.1pg/105 cells/24hours to 32.3 pg/105 cells/24hours) by XG1 cells was found in culture supernatant when SDF-1 was added in the culture system. The proliferation of myeloma cells induced by SDF-1 could be blocked by additional anti CXCR4 monoclonal antibody(12G5) or anti-IL-6 monoclonal antibody (B-E8); Moreover, we observed higher plasma levels of IL-6 in PB of 60% MM patients compared with those of healthy individuals. Finally, the levels of IL-6 were closely correlated with SDF-1 levels (y=0.8, P<0.01), These data indicated that in the IL-6-dependent myeloma cell lines or fresh myeloma samples and myeloma cell growth triggered by SDF-1 maybe due to up-regulation of autocrine and paracrine IL-6 by myeloma cells and stromal cells in BM. The results suggested that the expression of CXCR4 have an essential role in the proliferation and migration of myeloma cells in patients with multiple myeloma.4. Western-blot found that SDF-1 induced a transient up-regulation in the phophorylationof the p44/42 MAP kinase on XGs, but do not induced the phophorylation of the STATS on XGs. These study indicated that signal transduction of SDF-1/CXCR4 is through MAP kinase, but not STATS.5. We demonstrated that abnormal expression更多还原